In frame clonning adding a restriction site - (Oct/15/2008 )

Hi everybody,

I have a plasmid with a scAntibody cloned and a Myc-His Tag cloned downstream. I need to clone something in between but I am not too sure about the strategie. I need to clone my proteine in frame with the scAntibody, but it is a small protein and I have not restriction sites in the chain. I have thought to insert the restriction sites with the primers (I really need the restriction site because I think it is the only protocol I can use for the clonation), but I am not too sure how it will affect to the open reading frame. The questions are: how can I design the primers to minimize the number of pb added? and How will affect the insertion of some pb in my construct? Can I consider that it is an in frame clonation? (of course, I will maintain the open reading frame of my protein by always adding 3x pb)

thank you very very much!

To make sure I understand the situation, are you trying to clone your protein in-between the scAntibody and the myc-His tag?

If this is the case, the first thing you need to do is identify what restriction sites there are in between the scAntibody and myc-His tag. Then, design PCR primers that will amplify the protein you wish to clone in with the appropriate restriction sites at either end. You will have to check and see if everything will be in-frame after your ligation. It may be necessary to add 1 or 2 extra bp upstream and/or downstream of your protein of interest in order to achieve this.

It might also be a good idea to add a few extra small amino acids (eg. gly, ala) in-between your antibody and protein of interest to serve as a flexible linker that will allow the two proteins to fold correctly.

Ginger